The ability to spatially control activity of growth factors (GFs) is vital for more direct mimicry of complex developmental processes (e.g. organogenesis), and is particularly crucial for advancement of stem cell-based approaches to tissue regeneration. Our long-term goal is to develop approaches to spatially and temporally control GF presentation to stem cells in 3-D matrices, ultimately leading to tailored regeneration of lost or damaged tissues. Toward that end, this proposal describes a research program to develop 2-D cell culture substrates and 3-D matrices in which GF activity can be spatially controlled. We hypothesize that GFs that are immobilized specifically and reversibly to a material will retain biological activity, resulting in amplified signaling at specified sites. The proposal is divided into the following specific aims: Specific Aim 1 will develop and characterize a substrate for localized sequestering of two key developmental growth factors: IGF-1 and VEGF. The sequestering approach is based on surface presentation of peptide "handles" that interact specifically and reversibly with the growth factors of interest. Specific Aim 2 will systematically characterize growth factor bioactivity and will examine the ability of sequestered VEGF and IGF-1 to drive differentiation of adult human mesenchymal stem cells (MSCs) in culture. The affinity of peptide handles for soluble growth factor will be designed for optimal local growth factor activity, and the affinity will then be varied to achieve effective control over active growth factor concentration. This approach will be used to pattern growth factor activity, allowing for localized MSC differentiation. Specific Aim 3 will scale the approach developed in S.A.1 to a poly(ethylene glycol) (PEG) hydrogel matrix, in which the presence of peptide handles can be tightly controlled. This will, in turn, allow for control over the activity of sequestered VEGF in a 3-dimensional matrix. The approach to material synthesis will involve photocrosslinking of gels that contain polymer-peptide conjugates to allow for spatially distinct sequestering of growth factors. Specific Aim 4 will evaluate spatially controlled MSC differentiation within the PEG hydrogels developed in aim 3, focusing on differentiation of MSCs down the endothelial cell lineage. This aim is designed to demonstrate the utility of our approach in an application with broad impact on regenerative medicine and human health - engineering of intricate vascular networks within 3-D matrices. The results will serve as a springboard for development of an extensive research program in controlled GF presentation to control stem cell activity.